1. Field of the Invention
This invention relates to a method for the enhancement of serotonergic structures for the purpose of improving their visualization under microscopic examination by various techniques. This invention is particularly suitable for, but not limited, to the serotonergic raphe of the brainstem of nonhuman mammals.
2. Description of Prior Developments
For the understanding and, hence, rational treatment of neurologic and psychiatric diseases of serotonergic dysfunction it is desirable to be able to visualize in as much detail as possible the various manifestations of the structural elements in the serotonergic raphe of the hindbrain and the midbrain.
In vertebrates the midbrain is primarily responsible for the production and distribution of the neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) that is distributed to numerous forebrain structures involved in diverse behavioral and physiological functions. The dorsal raphe is the site of the majority of serotonergic cells thus far counted by immunocytochemical (alternately called immunohistochemical) techniques, and, hence, the primary source of serotonin in the central nervous system.
With traditional methodology, which has been used extensively, the serotonergic raphe is essentially similar within all, or most, species of vertebrates at numerous taxonomic levels. The raphe is phylogenetically old and conservative throughout phylogeny, testifing to its fundamental importance in vertebrates, including man.
To enhance the visualization of serotonergic cells in living nonhuman vertebrates, a common procedure is to pretreat the organism with L-tryptophan, the dietary amino acid precursor of the neurotransmitter serotonin. L-tryptophan is converted in two enzymatic steps to serotonin and the pretreatment is considered a method to increase the amount of neurotransmitter in the brain. It has been postulated that this is not a physiological treatment but a pharmacological one and is artifactual. Not all investigators pretreat with L-tryptophan.
All previous studies use a pretreatment time of from one to four or more hours prior to sacrificing the experimental animals. The rationale accepted in the prior art is that it takes considerable time for serotonin to be synthesized in the animal.
In conjunction with L-tryptophan pretreatment, a drug that blocks serotonin uptake at the synapse, hence preventing its catabolism, may be added to enhance the amount of neurotransmitter available for visualization.
The central importance of the dorsal raphe in the brainstem raphe in general is indicated by the quantity of neurotransmitter manufactured, number of serotonergic cells, and the amount and distribution of its efferent and afferent fibers.